Methods for generating stem cell-derived beta cells and methods of use thereof

ABSTRACT

Disclosed herein are methods for generating SC-β cells, and isolated populations of SC-β cells for use in various applications, such as cell therapy.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/975,457, filed Dec. 18, 2015, which claims the benefit of U.S.Provisional Application No. 62/093,974 filed on Dec. 18, 2014, theentire teachings of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Diabetes affects more than 300 million people worldwide according to theInternational Diabetes Federation. Type 1 diabetes and type 2 diabetesinvolve β cell destruction and/or β cell dysfunction. Diabetic patients,particularly those suffering from type 1 diabetes, could potentially becured through transplantation of β cells. While cadaveric human islettransplantation can render patients insulin independent for 5 years orlonger, such approach is limited due to the scarcity and quality ofdonor islets (Bellin et al., 2012). Generating an unlimited supply ofhuman β cells from stem cells could provide therapy to millions ofpatients as only a single cell type, the β cell, likely needs to beproduced, and the mode of delivery is well understood: transplantationto a vascularized location within the body with immunoprotection. Inaddition, screening to identify novel drugs that improve β cellfunction, survival, or proliferation is also delayed due to limitedislet supply and variability resulting from different causes of death,donor genetics, and other aspects in their isolation. As such, a steady,uniform supply of stem-cell-derived β cells would offer a useful drugdiscovery platform for diabetes. Moreover, genetically diversestem-cell-derived β cells could be used for disease modeling in vitro orin vivo.

SUMMARY OF THE INVENTION

There is a need for methods of generating stem cell-derived β (SC-(β)cells. The present invention is directed toward solutions to addressthis need, in addition to having other desirable characteristics.

In accordance with an embodiment of the present invention, a method forgenerating stem cell-derived β (SC-(β) cells is provided. The methodincludes contacting a cell population with agents to directdifferentiation to SC-β cells under conditions that promote cellclustering with additional agents comprising one or more of a) aneffective amount of an agent that decreases the level and/or activity ofrho-associated protein kinase (Rock) while the cell population comprisesi) FOXA2+, SOX2+ primitive foregut cells differentiating to PDX1+pancreatic progenitor cells, ii) PDX1+ pancreatic progenitor cellsdifferentiating into PDX1+, NKX6.1+ pancreatic progenitor cells, and/oriii) PDX1+, NKX6.1+ pancreatic progenitor cells differentiating tochromogranin A, NKX6.1+ endocrine progenitor cells; b) an effectiveamount of Activin A while the cell population comprises PDX1+ pancreaticprogenitor cells differentiating to PDX1+, NKX6.1+ pancreatic progenitorcells; and c) an effective amount of staurosporine while the cellpopulation comprises PDX1+, NKX6.1+ pancreatic progenitor cellsdifferentiating to chromogranin A, NKX6.1+ endocrine progenitor cells,thereby generating SC-β cells.

In accordance with aspects of the present invention, the method furthercomprises optionally washing the cell population after the PDX1+pancreatic progenitor cells have differentiated to PDX1+, NKX6.1+pancreatic progenitor cells to remove Activin A from contact with thecell population before contacting the cell population withstaurosporine.

In accordance with aspects of the present invention, the agent comprisesY-27632. In accordance with aspects of the present invention, theeffective amount of the agent comprises a concentration of 10 μM.

In accordance with aspects of the present invention, the effectiveamount of Activin A comprises a concentration of 5 ng/ml. In accordancewith aspects of the present invention, the effective amount ofstaurosporine comprises a concentration of 3 nM.

In accordance with aspects of the present invention, the cell populationcomprising PDX1+ pancreatic progenitor differentiating to PDX1+, NKX6.1+pancreatic progenitor cells is exposed to the agent and Activin A for aperiod of 6 days.

In accordance with aspects of the present invention, between at leastbetween 75% and 80% of the cell population differentiates tochromogranin A, NKX6.1+ endocrine progenitor cells beforedifferentiating to SC-β cells.

In accordance with aspects of the present invention, the agent i)prevents or minimizes cell cluster disintegration, ii) promotes cellsurvival, iii) increases cell cluster size, and combinations thereof.

In accordance with an embodiment of the present invention, an isolatedSC-β cell or population thereof that exhibits a glucose stimulatedinsulin secretion (GSIS) response both in vitro and in vivo is provided.In accordance with aspects of the present invention, the isolated SC-βcell or population thereof exhibits a stimulation index that is at leastbetween 1.0 and 3.0. In accordance with aspects of the presentinvention, the isolated SC-β cell or population thereof produces betweenapproximately 300 uIU and 4000 uIU per 30 minute incubation at a highglucose concentration.

In accordance with an embodiment of the present invention, amicrocapsule comprising the isolated SC-β cell or population thereofencapsulated therein is provided.

In accordance with an embodiment of the present invention, amacroencapsulation device comprising the isolated SC-β cell orpopulation thereof encapsulated therein is provided.

In accordance with an embodiment of the present invention, a cell linecomprising isolated SC-β cells that stably express insulin is provided.

In accordance with an embodiment of the present invention, assayscomprising the isolated SC-β cell, or population thereof, or the cellline are provided. The assays can be used for: i) identifying one ormore candidate agents which promote or inhibit a β cell fate selectedfrom the group consisting of β cell proliferation, β cell replication, βcell death, β cell function, β cell susceptibility to immune attack, andβ cell susceptibility to dedifferentiation or differentiation; or ii)identifying one or more candidate agents which promote thedifferentiation of at least one insulin-positive endocrine cell or aprecursor thereof into at least one SC-β cell.

In accordance with an embodiment of the present invention, a method forthe treatment of a subject in need thereof is disclosed. The methodincludes administering to a subject in need thereof i) an isolatedpopulation of SC-β cells, ii) a microcapsule comprising SC-β cellsencapsulated therein; and/or iii) a macroencapsulation device comprisingthe SC-β cells encapsulated therein. In accordance with an embodiment ofthe present invention, an isolated population of SC-β cells, amicrocapsule comprising the isolated population of SC-β cells, and/or amacroencapsulation device comprising the isolated population of SC-βcells is used for administering to a subject in need thereof. Inaccordance with aspects of the invention, the subject has, or has anincreased risk of developing diabetes or has, or has an increased riskof developing a metabolic disorder.

In accordance with an embodiment of the present invention, an artificialislet or pancreas comprising SC-β cells is provided.

The practice of the present invention will typically employ, unlessotherwise indicated, conventional techniques of cell biology, cellculture, molecular biology, transgenic biology, microbiology,recombinant nucleic acid (e.g., DNA) technology, immunology, and RNAinterference (RNAi) which are within the skill of the art. Non-limitingdescriptions of certain of these techniques are found in the followingpublications: Ausubel, F., et al., (eds.), Current Protocols inMolecular Biology, Current Protocols in Immunology, Current Protocols inProtein Science, and Current Protocols in Cell Biology, all John Wiley &Sons, N.Y., edition as of December 2008; Sambrook, Russell, andSambrook, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, 2001; Harlow, E. and Lane,D., Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, 1988; Freshney, R. I., “Culture of Animal Cells, AManual of Basic Technique”, 5th ed., John Wiley & Sons, Hoboken, N.J.,2005. Non-limiting information regarding therapeutic agents and humandiseases is found in Goodman and Gilman's The Pharmacological Basis ofTherapeutics, 11th Ed., McGraw Hill, 2005, Katzung, B. (ed.) Basic andClinical

Pharmacology, McGraw-Hill/Appleton & Lange; 10th ed. (2006) or 11thedition (July 2009). Non-limiting information regarding genes andgenetic disorders is found in McKusick, V.A.: Mendelian Inheritance inMan. A Catalog of Human Genes and Genetic Disorders. Baltimore: JohnsHopkins University Press, 1998 (12th edition) or the more recent onlinedatabase: Online Mendelian Inheritance in Man, OMIM™. McKusick-NathansInstitute of Genetic Medicine, Johns Hopkins University (Baltimore, Md.)and National Center for Biotechnology Information, National Library ofMedicine (Bethesda, Md.), as of May 1, 2010, World Wide Web URL:http://www.ncbi.nlm.nih.gov/omim/ and in Online Mendelian Inheritance inAnimals (OMIA), a database of genes, inherited disorders and traits inanimal species (other than human and mouse), athttp://omia.angis.org.au/contact.shtml. All patents, patentapplications, and other publications (e.g., scientific articles, books,websites, and databases) mentioned herein are incorporated by referencein their entirety. In case of a conflict between the specification andany of the incorporated references, the specification (including anyamendments thereof, which may be based on an incorporated reference),shall control. Standard art-accepted meanings of terms are used hereinunless indicated otherwise. Standard abbreviations for various terms areused herein.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other characteristics of the present invention will be morefully understood by reference to the following detailed description inconjunction with the attached drawings. The patent or application filecontains at least one drawing executed in color. Copies of this patentor patent application publication with color drawings will be providedby the Office upon request and payment of the necessary fee.

FIG. 1A and FIG. 1B are images of cells obtained using an exemplarycontrol protocol for generating SC-β cells and cells obtained using amethod for generating SC-β cells according to an embodiment of thepresent invention, respectively, demonstrating that the method forgenerating SC-β cells according to the present invention prevents orminimizes cell cluster disintegration, promotes cell survival, andincreases cell cluster size and number.

FIG. 2A are FACS plots demonstrating that the cells obtained at eachdifferentiation stage (S4, S5, and S6) using a method for generatingSC-β cells according to an embodiment of the present invention maintainmarker expression.

FIG. 2B are FACS plots of cells (e.g., SC-β cells) obtained using acontrol (e.g., DMSO) at the end of Step 6 (left panel) and cells (e.g.,SC-β cells) obtained at the end of Step 6 of method for generating SC-βcells according to an embodiment of the present invention (right panel),demonstrating that staurosporine treatment generates a near pureendocrine population.

FIG. 2C are FACS plots of cells (e.g., SC-β cells) obtained using acontrol (e.g., DMSO) at the end of Step 6 (left panel) and cells (e.g.,SC-β cells) obtained at the end of Step 6 of method for generating SC-βcells according to an embodiment of the present invention (right panel),demonstrating that staurosporine treatment generates a higher percentageof NKX6.1+, C-Peptide+cells.

FIG. 3A is a schematic illustrating six stages of differentiation ofhuman pluripotent stem cells to SC-β cells. hPSC=human pluripotent stemcell, DE=definitive endoderm cell, GT=gut tube cell, PP1=pancreaticprogenitor cell 1, PP2=pancreatic progenitor cell 2, EN=endocrineprogenitor cell, SC-β=stem cell-derived β cells.

FIG. 3B is a schematic illustrating an exemplary six stepdifferentiation protocol for generating SC-β cells from pluripotent stemcells, as described further in Pagliuca et al. 2014 and PCTInternational Application No. PCT/US2014/041992.

FIG. 4A is a schematic illustrating an exemplary method for generatingSC-β cells according to an embodiment of the present invention, e.g., bycontacting a cell population directed to differentiate to SC-β cellsunder conditions that promote cell clustering with an agent thatdecreases the level and/or activity of rho-associated protein kinase(Rock inhibitor), a TGF-β superfamily member (e.g., Activin A), and aprotein kinase inhibitor (e.g., staurosporine) during the stepsindicated.

FIG. 4B is a schematic illustrating an exemplary method for generatingSC-β cells according to an embodiment of the present invention, e.g., bycontacting a cell population directed to differentiate to SC-β cellsunder conditions that promote cell clustering with an agent thatdecreases the level and/or activity of rho-associated protein kinase(Rock inhibitor), a TGF-β superfamily member (e.g., Activin A), and aprotein kinase inhibitor (e.g., staurosporine) during the stepsindicated, and in which Step 4 is carried out for an additional 24 hourscompared to the method depicted in FIG. 4A.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to generating SC-β cells, inparticular SC-β cells that exhibit in vitro and in vivo function. Moreparticularly, work described herein demonstrates that contacting S2cells, S3 cells, and S4 cells during Step 3, Step 4 and Step 5 with aRock inhibitor with Activin A present during Step 4 in accordance withan embodiment of the present invention prevents or minimizes cellcluster disintegration, promotes cell survival, and increases the cellcluster size and number of cells in the resulting cell populations ascompared to a control, as is shown in FIG. 1A (control) and FIG. 1B.Additionally, work described herein demonstrates that contacting S4cells during Step 5 with staurosporine together with the Rock inhibitorand in the absence of Activin A generates cells that maintain markerexpression as shown in FIG. 2A, as well as generates a near pureendocrine population as shown in FIG. 2B, and a higher percentage ofNKX6.1+, C-Peptide+cells as shown in FIG. 2C. The SC-β cells generatedin accordance with the methods of the present invention can be used invarious assays to identify novel drugs that improve β cell function,survival, or proliferation, cell therapies (e.g., for treating diabetesand/or metabolic disorders), and in the construction of artificialislets and/or an artificial pancreas.

Some Definitions

“Differentiation” is the process by which an unspecialized(“uncommitted”) or less specialized cell acquires the features of aspecialized cell such as, for example, a pancreatic cell. Adifferentiated cell is one that has taken on a more specialized(“committed”) position within the lineage of a cell. The term“committed”, when applied to the process of differentiation, refers to acell that has proceeded in the differentiation pathway to a point where,under normal circumstances, it will continue to differentiate into aspecific cell type or subset of cell types, and cannot, under normalcircumstances, differentiate into a different cell type or revert to aless differentiated cell type. As used herein, the lineage of a celldefines the heredity of the cell, i.e., which cells it came from and towhat cells it can give rise. The lineage of a cell places the cellwithin a hereditary scheme of development and differentiation. Alineage-specific marker refers to a characteristic specificallyassociated with the phenotype of cells of a lineage of interest and canbe used to assess the differentiation of an uncommitted cell to thelineage of interest.

As used herein, “markers”, are nucleic acid or polypeptide moleculesthat are differentially expressed in a cell of interest. Differentialexpression means an increased level for a positive marker and adecreased level for a negative marker as compared to an undifferentiatedcell. The detectable level of the marker nucleic acid or polypeptide issufficiently higher or lower in the cells of interest compared to othercells, such that the cell of interest can be identified anddistinguished from other cells using any of a variety of methods knownin the art.

As used herein, a cell is “positive” or “+” for a specific marker (e.g.,expresses the marker) when the specific marker is sufficiently detectedin the cell. Similarly, the cell is “negative” or “−” for a specificmarker when the specific marker is not sufficiently detected in thecell. For example, positive by FACS is usually greater than 2%, whereasthe negative threshold by FACS is usually less than 1%.

The process of differentiating pluripotent stem cells into functionalpancreatic endocrine cells (i.e., SC-β cells) in vitro may be viewed insome aspects as progressing through six consecutive stages, as is shownin the exemplary protocol depicted in FIG. 3A. In this step-wiseprogression, “Stage 1” or “S1” refers to the first step in thedifferentiation process, the differentiation of pluripotent stem cellsinto cells expressing markers characteristic of definitive endodermcells (“DE”, “Stage 1 cells” or “S1 cells”). “Stage 2” refers to thesecond step, the differentiation of cells expressing markerscharacteristic of definitive endoderm cells into cells expressingmarkers characteristic of gut tube cells (“GT”, “Stage 2 cells” or “S2cells”). “Stage 3” refers to the third step, the differentiation ofcells expressing markers characteristic of gut tube cells into cellsexpressing markers characteristic of pancreatic progenitor 1 cells(“PP1”, “Stage 3 cells” or “S3 cells”). “Stage 4” refers to the fourthstep, the differentiation of cells expressing markers characteristic ofpancreatic progenitor 1 cells into cells expressing markerscharacteristic of pancreatic progenitor 2 cells (“PP2”, “Stage 4 cells”or “S4 cells”). “Stage 5” refers to the fifth step, the differentiationof cells expressing markers characteristic of pancreatic progenitor 2cells into cells expressing markers characteristic of pancreaticendoderm cells and/or pancreatic endocrine progenitor cells (“EN”,“Stage 5 cells” or “S5 cells”). “Stage 6” refers to the differentiationof cells expressing markers characteristic of pancreatic endocrineprogenitor cells into cells expressing markers characteristic ofpancreatic endocrine β cells (“SC-β cells”, “Stage 6 cells” or “S6cells”). It should be appreciated, however, that not all cells in aparticular population progress through these stages at the same rate,i.e., some cells may have progressed less, or more, down thedifferentiation pathway than the majority of cells present in thepopulation.

Characteristics of the various cell types associated with the stagesshown in FIG. 3A are now described. “Definitive endoderm cells,” as usedherein, refers to cells which bear the characteristics of cells arisingfrom the epiblast during gastrulation and which form thegastrointestinal tract and its derivatives. Definitive endoderm cellsexpress at least one of the following markers: FOXA2 (also known ashepatocyte nuclear factor 3β (“HNF3β”)), GATA4, SOX17, CXCR4, Brachyury,Cerberus, OTX2, goosecoid, C-Kit, CD99, and MIXL1. Markerscharacteristic of the definitive endoderm cells include CXCR4, FOXA2 andSOX17. Thus, definitive endoderm cells may be characterized by theirexpression of CXCR4, FOXA2 and SOX17. In addition, depending on thelength of time cells are allowed to remain in Stage 1, an increase inHNF4α may be observed.

“Gut tube cells,” as used herein, refers to cells derived fromdefinitive endoderm that can give rise to all endodermal organs, such aslungs, liver, pancreas, stomach, and intestine. Gut tube cells may becharacterized by their substantially increased expression of HNF4α overthat expressed by definitive endoderm cells. For example, a ten to fortyfold increase in mRNA expression of HNF4α may be observed during Stage2.

“Pancreatic progenitor 1 cells,” as used herein, refers to endodermcells that give rise to the esophagus, lungs, stomach, liver, pancreas,gall bladder, and a portion of the duodenum. Pancreatic progenitor 1cells express at least one of the following markers: PDX1, FOXA2, CDX2,SOX2, and HNF4α. Pancreatic progenitor 1 cells may be characterized byan increase in expression of PDX1, compared to gut tube cells. Forexample, greater than fifty percent of the cells in Stage 3 culturestypically express PDX1.

“Pancreatic progenitor 2 cells,” as used herein, refers to cells thatexpress at least one of the following markers: PDX1, NKX6.1, HNF6, NGN3,SOX9, PAX4, PAX6, ISL1, gastrin, FOXA2, PTF1a, PROX1 and HNF4α.Pancreatic progenitor 2 cells may be characterized as positive for theexpression of PDX1, NKX6.1, and SOX9.

“Pancreatic endocrine progenitor cells” or “endocrine progenitor cells”are used interchangeably herein to refer to pancreatic endoderm cellscapable of becoming a pancreatic hormone expressing cell. Pancreaticendocrine progenitor cells express at least one of the followingmarkers: NGN3; NKX2.2; NeuroD1; ISL1; PAX4; PAX6; or ARX. Pancreaticendocrine progenitor cells may be characterized by their expression ofNKX2.2 and NeuroD1.

A “precursor thereof” as the term relates to a pancreatic endocrineprogenitor cell refers to any cell that is capable of differentiatinginto a pancreatic endocrine progenitor cell, including for example, apluripotent stem cell, a definitive endoderm cell, a gut tube cell, or apancreatic progenitor cell, when cultured under conditions suitable fordifferentiating the precursor cell into the pancreatic pro endocrinecell.

“Pancreatic endocrine cells,” as used herein, refer to cells capable ofexpressing at least one of the following hormones: insulin, glucagon,somatostatin, ghrelin, and pancreatic polypeptide. In addition to thesehormones, markers characteristic of pancreatic endocrine cells includeone or more of NGN3, NeuroD1, ISL1, PDX1, NKX6.1, PAX4, ARX, NKX2.2, andPAX6. Pancreatic endocrine cells expressing markers characteristic of βcells can be characterized by their expression of insulin and at leastone of the following transcription factors: PDX1, NKX2.2, NKX6.1,NeuroD1, ISL1, HNF30, MAFA and PAX6.

The terms “stem cell-derived β cell” and “SC-β cell” are usedinterchangeably herein to refer to non-native cells differentiated invitro (e.g., from pluripotent stem cells) that display at least onemarker indicative of a pancreatic β cell (e.g., PDX-1 or NKX6-1),expresses insulin, and display a GSIS response characteristic of anendogenous mature β cell both in vitro and in vivo. The GSIS response ofthe SC-β cells can be observed within two weeks of transplantation ofthe SC-β cell into a host (e.g., a human or animal). It is to beunderstood that the SC-β cells need not be derived (e.g., directly) fromstem cells, as the methods of the disclosure are capable of derivingSC-β cells from any endocrine progenitor cell that expresses insulin orprecursor thereof using any cell as a starting point (e.g., one can useembryonic stem cells, induced-pluripotent stem cells, progenitor cells,partially reprogrammed somatic cells (e.g., a somatic cell which hasbeen partially reprogrammed to an intermediate state between an inducedpluripotent stem cell and the somatic cell from which it was derived),multipotent cells, totipotent cells, a transdifferentiated version ofany of the foregoing cells, etc, as the invention is not intended to belimited in this manner). In some aspects, human cells are excluded thatare derived from human embryonic stem cells obtained exclusively by amethod necessitating the destruction of an embryo. The skilled artisanis well aware of such methods and how to avoid them for the purposes ofgenerating SC-β cells according to the methods of the present invention.

Used interchangeably herein are “d1”, “1d”, and “day 1”; “d2”, “2d”, and“day 2”, etc. These number letter combinations refer to a specific dayof incubation in the different stages during the stepwisedifferentiation protocol of the instant application.

Methods for Generating SC-β Cells

Recently, two protocols for directing the differentiation of pluripotentstem cells into insulin-producing endocrine cells that express keymarkers of mature pancreatic β cells (e.g., SC-β cells) have beenreported, each of which includes differentiating cells into endocrineprogenitor cells that can be directed to differentiate into SC-β cells,as well as protocols for directing the pancreatic endocrine progenitorcells into SC-β cells, which can be used in the method disclosed hereinfor generating SC-β cells. First, as is shown in FIG. 3B, a six-stageprotocol for the large-scale production of functional human β cellsusing human pluripotent stem cells (hPSC) by sequential modulation ofmultiple signaling pathways in a three-dimensional cell culture system,without using any transgenes or genetic modification, was used togenerate glucose-responsive, monohormonal insulin-producing cells thatexhibited key β cell markers and β cell ultrastructure (see Pagliuca etal., 2014 and PCT International Application No. PCT/US2014/041992, bothof which are incorporated herein by reference in their entirety).Pagliuca and colleagues reported that such cells mimicked the functionof human islets in vitro and in vivo, and demonstrated the potentialutility of such cells for in vivo transplantation to treat diabetes.Secondly, a seven-stage protocol that converts human embryonic stemcells (hESCs) into insulin-producing cells that expressed key markers ofmature pancreatic β cells, such as MAFA, and displayedglucose-stimulated insulin secretion like that of human islets usingstatic incubations in vitro was described (Rezania et al., 2014). Cellsproduced by such protocol, referred to as S7 cells, were found torapidly reverse diabetics in mice within a little over a month.

FIG. 4A shows an exemplary method for generating SC-β cells inaccordance with an embodiment of the present invention that results inlarger cell clusters comprising greater numbers of SC-β cells exhibitinga more pure endocrine population and greater number of SC-β cellsexhibiting co-expression of NKX6.1 and C-peptide.

In accordance with an example embodiment of the present invention, amethod for generating stem cell-derived β (SC-(β) cells comprisescontacting a cell population directed to differentiate to SC-β cellsunder conditions that promote cell clustering with a) an effectiveamount of an agent that decreases the level and/or activity ofrho-associated protein kinase (Rock) while the cell population comprisesi) FOXA2+, SOX2+ primitive foregut cells differentiating to PDX1+pancreatic progenitor cells, ii) PDX1+ pancreatic progenitor cellsdifferentiating into PDX1+, NKX6.1+ pancreatic progenitor cells, and/oriii) PDX1+, NKX6.1+ pancreatic progenitor cells differentiating tochromogranin A, NKX6.1+ endocrine progenitor cells; b) an effectiveamount of a TGF-β superfamily member while the cell population comprisesii) PDX1+ pancreatic progenitor cells differentiating to PDX1+, NKX6.1+pancreatic progenitor cells; and c) an effective amount of a proteinkinase inhibitor while the cell population comprises iii) PDX1+, NKX6.1+pancreatic progenitor cells differentiating to chromogranin A, NKX6.1+endocrine progenitor cells, thereby generating SC-β cells.

“Contacting”, “contacting the cell” and any derivations thereof as usedherein, refers to any means of introducing an agent (e.g., nucleicacids, peptides, ribozymes, antibodies, small molecules, etc.) into atarget cell or an environment in which the cell is present (e.g., cellculture), including chemical and physical means, whether directly orindirectly. Contacting also is intended to encompass methods of exposinga cell, delivering to a cell, or ‘loading’ a cell with an agent by viralor non-viral vectors, and wherein such agent is bioactive upon delivery.The method of delivery will be chosen for the particular agent and use.Parameters that affect delivery, as is known in the medical art, caninclude, inter alia, the cell type affected, and cellular location. Insome aspects, contacting includes administering the agent to a subject.In some aspects, contacting refers to exposing a cell or an environmentin which the cell is located (e.g., cell culture medium) to at least oneagent that decreases the level and/or activity of Rock (also referred toherein as “ROCK inhibitor”). In some aspects, contacting refers toexposing a cell or an environment in which the cell is located (e.g.,cell culture medium) to a TGF-β superfamily member (e.g., Activin A). Insome aspects, contacting refers to exposing a cell or an environment inwhich the cell is located (e.g., cell culture medium) to a proteinkinase inhibitor (e.g., staurosporine).

In accordance with aspects of the present invention, the method includesan optional step of washing the cell population after the PDX1+pancreatic progenitor cells have differentiated to PDX1+, NKX6.1+pancreatic progenitor cells to remove Activin A from contact with thecell population before contacting the cell population withstaurosporine.

The methods of the present invention contemplate: i) contacting cells orcell populations (e.g., comprising S2, S3, and S4 cells) with aneffective amount of one or more Rock inhibitors; ii) contacting cells orcell populations (e.g., comprising S3 cells) with an effective amount ofa TGF-β superfamily member (e.g., Activin A); and iii) contacting cellsor cell populations (e.g., comprising S4 cells) with an effective amountof a protein kinase inhibitor (e.g., staurosporine). An “effectiveamount” of an agent (or composition containing such agent) refers to theamount sufficient to achieve a desired effect, e.g., when delivered to acell or subject according to a selected administration form, route,and/or schedule. As will be appreciated by those of ordinary skill inthis art, the absolute amount of a particular agent or composition thatis effective may vary depending on such factors as the desiredbiological or pharmacological endpoint, the agent to be delivered, thetarget tissue, etc. Those of ordinary skill in the art will furtherunderstand that an “effective amount” may be contacted with cells oradministered in a single dose, or the desired effect may be achieved byuse of multiple doses. An effective amount of a composition may be anamount sufficient to reduce the severity of or prevent one or moresymptoms or signs of a disorder (e.g., diabetes). In some aspects, theeffective amount of the agent that decreases the level and/or activityof ROCK comprises a concentration of between about 0.1 μM and about 110μM. In some aspects, the effective amount of the agent (e.g., ROCKinhibitor) comprises 10 μM. In some aspects, the effective amount of theTGF-β superfamily member comprises a concentration of between about 1ng/ml to about 1000 ng/ml. In some aspects, the effective amount of theTGF-β superfamily member comprises 5 ng/ml. In some aspects, theeffective amount of the protein kinase inhibitor comprises aconcentration of between about 0.1 nM and 110 nM. In some aspects, theeffective amount of the protein kinase inhibitor comprises aconcentration of 3nM.

The present invention contemplates using any agent that decreases thelevel and/or activity of ROCK in the methods for generating SC-β cells,e.g., ROCK inhibitors can be small organic or inorganic molecules;saccharides; oligosaccharides; polysaccharides; biologicalmacromolecules, e.g., peptides, proteins, and peptide analogs andderivatives; peptidomimetics; nucleic acids and nucleic acid analogs andderivatives (including but not limited to microRNAs, siRNAs, shRNAs,antisense RNAs, a ribozymes, and aptamers); an extract made frombiological materials such as bacteria, plants, fungi, or animal cells;animal tissues; naturally occurring or synthetic compositions; and anycombinations thereof.

Exemplary ROCK inhibitors include, but are not limited to a smallorganic molecule ROCK inhibitor selected from the group consisting ofN-[(1S)-2-Hydroxy-1-phenylethyl]-N′-[4-(4-pyridinyl)phenyl]-urea(AS1892802), fasudil hydrochloride (also known as HA 1077),-[3-[[2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-6-yl]oxy]phenyl]-4-[2-(4-morpholinyl)ethoxy]benzamide(GSK269962),4-[4-(Trifluoromethyl)phenyl]-N-(6-Fluoro-1H-indazol-5-yl)-2-methyl-6-oxo-1,4,5,6-tetrahydro-3-pyridinecarboxamide(GSK 429286),(S)-(+)-2-Methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepinedihydrochloride (H 1152 dihydrochloride),(S)-(+)-4-Glycyl-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepinedihydrochloride (glycyl-H 1152 dihydrochloride),N-[(3-Hydroxyphenyl)methyl]-N′-[4-(4-pyridinyl)-2-thiazolyl]ureadihydrochloride (RKI 1447 dihydrochloride),(3S)-1-[[2-(4-Amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4,5-c]pyridin-7-yl]carbonyl]-3-pyrrolidinaminedihydrochloride (SB772077B dihydrochloride),N-[2-[2-(Dimethylamino)ethoxy]-4-(1H-pyrazol-4-yl)phenyl-2,3-dihydro-1,4-benzodioxin-2-carboxamidedihydrochloride (SR 3677 dihydrochloride), andtrans-4-[(1R)-1-Aminoethyl]-N-4-pyridinylcyclohexanecarboxamidedihydrochloride (Y-27632 dihydrochloride),N-Benzyl[2-(pyrimidin-4-yl)amino]thiazole-4-carboxamide (Thiazovivin),Rock Inhibitor, a isoquinolinesulfonamide compound (Rho KinaseInhibitor), N-(4-Pyridyl)-N′-(2,4,6-trichlorophenyl)urea (Rho KinaseInhibitor II), 3-(4-Pyridyl)-1H-indole (Rho Kinase Inhibitor III,Rockout), and 4-pyrazoleboronic acid pinacol ester; a Rock antibodycommercially available from Santa Cruz Biotechnology selected from thegroup consisting of Rock-1 (B1), Rock-1 (C-19), Rock-1 (H-11), Rock-1(G-6), Rock-1 (H-85), Rock-1 (K-18), Rock-2 (C-20), Rock-2 (D-2), Rock-2(D-11), Rock-2 (N-19), Rock-2 (H-85), Rock-2 (30-J); a ROCK CRISPR/Cas9knockout plasmid selected from the group consisting of Rock-1CRISPR/Cas9 KO plasmid (h), Rock-2

CRISPR/Cas9 KO plasmid (h), Rock-1 CRISPR/Cas9 KO plasmid (m), Rock-2CRISPR/Cas9 KO plasmid (m); a ROCK siRNA, shRNA plasmid and/or shRNAlentiviral particle gene silencer selected from the group consisting ofRock-1 siRNA (h): sc-29473, Rock-1 siRNA (m): sc-36432, Rock-1 siRNA(r): sc-72179, Rock-2 siRNA (h): sc-29474, Rock-2 siRNA (m): sc-36433,Rock-2 siRNA (r): sc-108088.

In some aspects, a ROCK inhibitor decreases the level and/or activity ofROCK in cells contacted by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, or 95% relative to the level or activity of ROCK in thecells in the absence of contact with the ROCK inhibitor. While notrequired, a ROCK inhibitor can completely inhibit the level and/oractivity of ROCK in the cells. It should be appreciated that the ROCKinhibitors may decrease the level and/or activity of ROCK in any cell inthe population in which the S2, S3 and/or S4 cells are differentiating,including endocrine progenitor cells in population, SC-β cells, and anyprecursors thereof.

In accordance with aspects of the present invention, the agent (e.g.,ROCK inhibitor) comprises Y-27632.

An effective amount of Y-27632 for use in the methods of the presentinvention can be, for example, between about 0.1 μM and about 110 μM. Insome aspects, an effective amount of tY-27632 comprises 10 μM.

The present invention contemplates using any agent TGF-β superfamilymember. Exemplary such members include, but are not limited to, Nodal,Activin A, Activin B, bone morphogenic protein-2 (BMP2), bonemorphogenic protein-4 (BMP4) and functional fragments thereof. Inaccordance with aspects of the present invention, the TGF-β superfamilymember comprises Activin A.

An effective amount of Activin A for use in the methods of the presentinvention can be, for example, between about 1 ng/ml to about 1000ng/ml. In accordance with aspects of the present invention, an effectiveamount of Activin A comprises a concentration of 5 ng/ml.

The present invention contemplates using any protein kinase inhibitorthat is capable of generating a near pure endocrine population orincreasing the number of cells in the population coexpressing andNKX6.1+, C-peptide in the methods for generating SC-β cells.

In accordance with aspects of the present invention, the protein kinaseinhibitor comprises staurosporine. An effective amount of staurosporinefor use in the methods of the present invention can be, for example,between about 0.1 nM and about 110 nM. In accordance with aspects of thepresent invention, an effective amount of staurosporine comprises aconcentration of 3 nM.

In accordance with aspects of the present invention, as is shown in FIG.4B, the cell population comprising ii) PDX1+ pancreatic progenitordifferentiating to PDX1+, NKX6.1+ pancreatic progenitor cells is exposedto the agent and TGF-β superfamily member (e.g., Activin A) for a periodof 6 days.

Preferably, a maximum number of cells in the population differentiate tochromogranin A, NKX6.1+ endocrine progenitor cells beforedifferentiating into SC-β cells. The number of cells in the populationdifferentiating to chromogranin A, NKX6.1+ endocrine progenitor cellsbefore differentiating into SC-β cells is 5%, 10%, 15%, 20%, 25%, 30%,40%, 45%, 50%, 55%, 60%, 65%, 70%, or more. In accordance with aspectsof the present invention, between at least between 75% and 80% of thecell population differentiates to chromogranin A, NKX6.1+ endocrineprogenitor cells before differentiating to SC-β cells.

SC-β Cells Obtained by the Method of Generating SC-β Cells

In accordance with an embodiment of the present invention, an isolatedSC-β cell or population thereof generated according to a methoddescribed herein is provided. The isolated SC-β cell or populationexhibits a GSIS response both in vitro and in vivo. The isolated SC-βcell or population also exhibits at least one characteristic feature ofa mature endogenous β cell (e.g., monohormonality). In some aspects, anisolated SC-β cell or population thereof exhibits a stimulation index ofbetween about 1.0 and about 3.0. In some aspects, an isolated SC-β cellor population thereof produces between approximately 300 uIU to about4000 uIU per 30 minute per 10⁶ total cells incubation at a high glucoseconcentration.

The SC-β cells disclosed herein share many distinguishing features ofnative f3 cells, but are different in certain aspects (e.g., geneexpression profiles). In some embodiments, the SC-β cell is non-native.As used herein, “non-native” means that the SC-β cells are markedlydifferent in certain aspects from β cells which exist in nature, i.e.,native β cells. It should be appreciated, however, that these markeddifferences typically pertain to structural features which may result inthe SC-β cells exhibiting certain functional differences, e.g., althoughthe gene expression patterns of SC-β cells differs from native β cells,the SC-β cells behave in a similar manner to native β cells but certainfunctions may be altered (e.g., improved) compared to native β cells.For example, a higher frequency of SC-β cells respond to 20 mM glucosecompared to the frequency of native β cells. Other differences betweenSC-β cells and native β cells would be apparent to the skilled artisanbased on the data disclosed herein.

The SC-β cells of the disclosure share many characteristic features of βcells which are important for normal β cell function. For example, theSC-β cells (e.g., human) generated according to the methods describedherein may further exhibit at least one of the following characteristicsof an endogenous mature pancreatic β cell: i) a response to multipleglucose challenges that resembles the response of endogenous islets(e.g., at least one, at least two, or at least three or more sequentialglucose challenges); ii) a morphology that resembles the morphology ofan endogenous β cell; iii) packaging of insulin into secretory granulesor encapsulated crystalline insulin granules; iv) a stimulation index ofgreater than at least 1.4; v) cytokine-induced apoptosis in response tocytokines; vi) enhanced insulin secretion in response to knownantidiabetic drugs (e.g., secretagogues); vii) monohormonal, i.e., theydo not abnormally co-express other hormones, such as glucagon,somatostatin or pancreatic polypeptide; viii) a low rate of replication;and ix) increased intracellular Ca²⁺ in response to glucose.

In accordance with an embodiment of the present invention, amicrocapsule comprising the isolated SC-β cell or population thereofencapsulated therein is provided.

In accordance with an embodiment of the present invention, amacroencapsulation device comprising the isolated SC-β cell orpopulation thereof is provided.

In accordance with an embodiment of the present invention, a cell linecomprising an isolated SC-β cell that stably expresses insulin isprovided.

Assays

In accordance with an embodiment of the present invention, an isolatedSC-β cell or population thereof generated according to the methodsherein, or an SC-β cell that stably expresses insulin, can be used invarious assays. In some aspects, an isolated SC-β cell, populationthereof, or an SC-β cell that stably expresses insulin, can be used inan assay to identify one or more candidate agents which promote orinhibit a β cell fate selected from the group consisting of β cellproliferation, β cell replication, β cell death, (3 cell function, βcell susceptibility to immune attack, and β cell susceptibility todedifferentiation or differentiation. In some aspects, an isolated SC-βcell, population thereof, or an SC-β cell that stably expresses insulin,can be used in an assay to identify one or more candidate agents whichpromote the differentiation of at least one insulin-positive endocrinecell or a precursor thereof into at least one SC-β cell. The assaystypically involve contacting the isolated SC-β cell, population thereof,or an SC-β cell that stably expresses insulin, with one or morecandidate agents to be assessed for its ability to i) promote or inhibita β cell fate selected from the group consisting of β cellproliferation, β cell replication, β cell death, β cell function, β cellsusceptibility to immune attack, and β cell susceptibility todedifferentiation or differentiation, or ii) promoting thedifferentiation of at least one insulin-positive endocrine cell or aprecursor thereof into at least one SC-β cell and assessing whether thecandidate agent possesses the ability to i) promote or inhibit a β cellfate selected from the group consisting of β cell proliferation, β cellreplication, β cell death, β cell function, β cell susceptibility toimmune attack, and β cell susceptibility to dedifferentiation ordifferentiation, or ii) promoting the differentiation of at least oneinsulin-positive endocrine cell or a precursor thereof into at least oneSC-β cell.

Methods for Treatment

In accordance with an embodiment of the present invention, methods forthe treatment of a subject in need thereof are provided. The methodsentail administering to a subject in need thereof an isolated populationof SC-β cells or a microcapsule comprising SC-β cells encapsulatedtherein. In some aspects, the subject is in need of additional β cells.In some aspects, the subject has, or has an increased risk of developingdiabetes. A SC-β cell or population (e.g., isolated) of SC-β cellsgenerated by a method of the present invention can be administered to asubject for treatment of type 1 or type 2 diabetes. In some aspects, thesubject has, or has an increased risk of developing, a metabolicdisorder. In some aspects, administering to the subject comprisesimplanting SC-β cells, a microcapsule comprising SC-β cells, or amacroencapsulation device comprising SC-β cells into the subject. Thesubject may be a human subject or an animal subject. In some aspects,the cells may be implanted as dispersed cells or formed into clustersthat may be infused into the hepatic portal vein. In some aspects, cellsmay be provided in biocompatible degradable polymeric supports, porousnon-degradable devices or encapsulated to protect from host immuneresponse. Cells may be implanted into an appropriate site in arecipient. The implantation sites include, for example, the liver,natural pancreas, renal subcapsular space, omentum, peritoneum,subserosal space, intestine, stomach, or a subcutaneous pocket.

To enhance further differentiation, survival or activity of theimplanted cells in vivo, additional factors, such as growth factors,antioxidants or anti-inflammatory agents, can be administered before,simultaneously with, or after the administration of the cells. Thesefactors can be secreted by endogenous cells and exposed to theadministered cells in situ. Implanted cells can be induced todifferentiate by any combination of endogenous and exogenouslyadministered growth factors known in the art.

The amount of cells used in implantation depends on a number of variousfactors including the patient's condition and response to the therapy,and can be determined by one skilled in the art.

In some aspects, the method of treatment further comprises incorporatingthe cells into a three-dimensional support prior to implantation. Thecells can be maintained in vitro on this support prior to implantationinto the patient. Alternatively, the support containing the cells can bedirectly implanted in the patient without additional in vitro culturing.The support can optionally be incorporated with at least onepharmaceutical agent that facilitates the survival and function of thetransplanted cells.

Artificial Islet or Pancreas

In accordance with an embodiment of the present invention, an artificialislet or pancreas is provided. The artificial islet or pancreas can beconstructed using the SC-β cells generated according to the methodsdescribed herein.

An artificial pancreas is a device that encapsulates and nurtures isletsof Langerhans to replace the islets and β cells destroyed by type 1diabetes. An artificial pancreas may contain a million islets or more,and may be implanted in the peritoneal cavity or under the skin where itcan respond to changing blood glucose levels by releasing hormones, suchas insulin. An artificial pancreas may be made using living (e.g.,glucose-sensing and insulin secreting islets) and nonliving components(e.g., to shield the islets from the diabetic's body and its destructiveimmune mechanism while permitting the islets to thrive).

The present invention contemplates using β cells in any artificialpancreas. In some aspects, the artificial pancreas comprisesmicroencapsulated or coated islets comprising SC-β cells generatedaccording to the methods herein. In some aspects, the artificialpancreas comprises a macroencapsulation device into which islet cellscomprising SC-β cells generated according to the methods herein aregrouped together and encapsulated. In some aspects, themacroencapsulation device comprises a PVA hydrogel sheet for anartificial pancreas of the present invention (Qi et al., 2004). In someaspects, the artificial islet comprises SC-β cells generated accordingto the methods herein, along with other islet cells (α, δ, etc.) in theform of an islet sheet. The islet sheet comprises a layer of artificialhuman islets comprising the SC-β cells macroencapsulated within amembrane (e.g., of ultra-pure alginate). The sheet membrane isreinforced with mesh and may be coated on the surface to prevent orminimize contact between the cells encapsulated inside and thetransplantation recipient's host immune response. Oxygen, glucose, andother nutrients readily diffuse into the sheet through the membranenurturing the islets, and hormones, such as insulin readily diffuse out.Additional examples of membranes designed formacroencapsulation/implantation of an artificial islet or pancreas canbe found in the literature (Isayeva et al. 2003). Another example of amacroencapsulated implant suitable for an artificial islet or pancreascan be found in the literature (Aurélien, et al. 2014).

Terminology

The articles “a”, “an” and “the” as used herein, unless clearlyindicated to the contrary, should be understood to include the pluralreferents. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. It should it be understood that,in general, where the invention, or aspects of the invention, is/arereferred to as comprising particular elements, features, etc., certainembodiments of the invention or aspects of the invention consist, orconsist essentially of, such elements, features, etc. For purposes ofsimplicity those embodiments have not in every case been specificallyset forth in haec verba herein. It should also be understood that anyembodiment of the invention, e.g., any embodiment found within the priorart, can be explicitly excluded from the claims, regardless of whetherthe specific exclusion is recited in the specification. For example, anyagent may be excluded from the genera of ROCK inhibitors, TGF-βsuperfamily members, and protein kinase inhibitors.

Where ranges are given herein, the invention includes embodiments inwhich the endpoints are included, embodiments in which both endpointsare excluded, and embodiments in which one endpoint is included and theother is excluded. It should be assumed that both endpoints are includedunless indicated otherwise. Furthermore, it is to be understood thatunless otherwise indicated or otherwise evident from the context andunderstanding of one of skill in the art, values that are expressed asranges can assume any specific value or subrange within the statedranges in different embodiments of the invention, to the tenth of theunit of the lower limit of the range, unless the context clearlydictates otherwise. It is also understood that where a series ofnumerical values is stated herein, the invention includes embodimentsthat relate analogously to any intervening value or range defined by anytwo values in the series, and that the lowest value may be taken as aminimum and the greatest value may be taken as a maximum. Numericalvalues, as used herein, include values expressed as percentages. For anyembodiment of the invention in which a numerical value is prefaced by“about” or “approximately”, the invention includes an embodiment inwhich the exact value is recited. For any embodiment of the invention inwhich a numerical value is not prefaced by “about” or “approximately”,the invention includes an embodiment in which the value is prefaced by“about” or “approximately”. “Approximately” or “about” generallyincludes numbers that fall within a range of 1% or in some embodiments5% of a number in either direction (greater than or less than thenumber) unless otherwise stated or otherwise evident from the context(except where such number would impermissibly exceed 100% of a possiblevalue).

Furthermore, it is to be understood that the invention encompasses allvariations, combinations, and permutations in which one or morelimitations, elements, clauses, descriptive terms, etc., from one ormore of the listed claims is introduced into another claim dependent onthe same base claim (or, as relevant, any other claim) unless otherwiseindicated or unless it would be evident to one of ordinary skill in theart that a contradiction or inconsistency would arise. Where elementsare presented as lists, e.g., in Markush group or similar format, it isto be understood that each subgroup of the elements is also disclosed,and any element(s) can be removed from the group.

Certain claims are presented in dependent form for the sake ofconvenience, but any dependent claim may be rewritten in independentformat to include the limitations of the independent claim and any otherclaim(s) on which such claim depends, and such rewritten claim is to beconsidered equivalent in all respects to the dependent claim (eitheramended or unamended) prior to being rewritten in independent format. Itshould also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one act,the order of the acts of the method is not necessarily limited to theorder in which the acts of the method are recited, but the inventionincludes embodiments in which the order is so limited. It iscontemplated that all aspects described above are applicable to alldifferent embodiments of the invention. It is also contemplated that anyof the above embodiments can be freely combined with one or more othersuch embodiments whenever appropriate.

REFERENCES

1. Bellin et al., (2012). Potent induction immunotherapy promoteslong-term insulin independence after islet transplantation in type 1diabetes. Am. J. Transplant. 12, 1576-1583.

2. Pagliuca et al. (2014). Generation of Functional Human Pancreatic βcells In Vitro. Cell. 159, 428-439.

3. Rezania et al. (2014). Reversal of diabetes with insulin-producingcells derived in vitro from human pluripotent stem cells. Nat. Biotech.32(11), 1121-1133.

4. Isayeva, et al. (2003). Characterization and performance of membranesdesigned for macroencapsulation/implantation of pancreatic islet cells.Biomaterials 24(20), 3483-3491.

5. Motte, et al. (2014). Composition and function of macroencapsulatedhuman embryonic stem cell-derived implants: comparison with clinicalhuman islet cell grafts. American Journal of Physiology-Endocrinologyand Metabolism 307(9), E838-E846.

6. Qi et al. (2004). PVA hydrogel sheet macroencapsulation of thebioartificial pancreas. Biomaterials 24(27), 5885-5892

What is claimed is:
 1. A composition comprising a rho-associated proteinkinase inhibitor, staurosporine, and a population of pancreaticprogenitor cells that express PDX1 and NKX6.1.
 2. The composition ofclaim 1, wherein said rho-associated protein kinase inhibitor isselected from the group consisting of: fasudil hydrochloride, GSK269962,GSK 429286, Y-27632, and Thiazovivin.
 3. The composition of claim 1,wherein said rho-associated protein kinase inhibitor has a concentrationof between about 0.1 μM and about 110 μM in said composition.
 4. Thecomposition of claim 1, wherein said population of pancreatic progenitorcells are not genetically modified.
 5. The composition of claim 1,wherein said staurosporine has a concentration of between about 0.1 nMand about 110 nM in said composition.
 6. The composition of claim 1,wherein the composition further comprises XXI.
 7. The composition ofclaim 6, wherein the composition further comprises SANT1.
 8. Thecomposition of claim 7, wherein the composition further comprisesretinoic acid (RA).
 9. The composition of claim 1, wherein thecomposition further comprises SANT1.
 10. The composition of claim 1,wherein the composition further comprises retinoic acid (RA).
 11. Thecomposition of claim 1, wherein the composition further comprisesbetacellulin.
 12. The composition of claim 1, wherein the compositionfurther comprises T3.